Vatchavai Bhaskara Raju et al, Asian Journal of Pharmaceutical Technology & Innovation, 05 (23); 2017; 07-12

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Asian Journal of Pharmaceutical Technology & Innovation ISSN: 2347-8810 Research Article Received on: 26-02-2017 Accepted on: 11-03-2017 Published on: 15-04-2017 Corresponding Author: * Dr. V.

1 Asian Journal of Pharmaceutical Technology & Innovation ISSN: Research Article Received on: Accepted on: Published on: Corresponding Author: * Dr. V. Bhaskara Raju, Associate Professor, Sri Vasavi Institute of Pharmaceutical Sciences. Pedatadepalli, Tadepalligudem, West Godavari, , Andhra Pradesh. Development and Validation of New RP-HPLC Method for the Estimation of Dasatinib in Pharmaceutical Dosage Forms Vatchavai Bhaskara Raju*, Bonthu Mohan Gandhi, Kamatham Srinivas Sumanth, Kolli Srinivas, Badarala Pallavi ABSTRACT A simple, precise, accurate RP-HPLC method was developed and validated for the estimation of Dasatinib in pharmaceutical dosage forms. An Cosmicsil BDS C18 column (150 mm x 4.6 mm), 5 µ particle size was used as stationary phase with mobile phase consisting of Phosphate buffer: a mixture of acetonitrile and methanol in the ratio of 50:50, v/v. The flow rate was maintained at 1 ml/min and effluents were monitored at 315 nm. The retention time was min. The linearity of the method was observed in the concentration range of µg/ml with correlation coefficient of The percentage assay of Dasatinib was %. The method was validated for its accuracy, precision and system suitability. The results obtained in the study were within the limits of ICH guidelines and hence this method can be used for the estimation of Dasatinib in pharmaceutical dosage forms. * Idbhaskar_pharmaanalyst@yahoo.co.in Key-words: Dasatinib, Cosmicsil BDS C18 column, RP-HPLC, Validation. Cite this article as: Vatchavai Bhaskara Raju, Bonthu Mohan Gandhi, Kamatham Srinivas Sumanth, Kolli Srinivas, Badarala Pallavi, Development and Validation of New RP-HPLC Method for the Estimation of Dasatinib in Pharmaceutical Dosage Forms, Asian Journal of Pharmaceutical Technology & Innovation, 05 (23); 07-12, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem , Andhra Pradesh 7

2 INTRODUCTION: Dasatinib (Fig. 1) is chemically N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2- methylpyrimidin-4-yl]amino]-1,3-thiazole-5-carboxamide (1). Dasatinib is an oral dual BCR/ABL and SRC family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia (2). Literature survey revealed that few analytical methods such as LC-MS 3-8, UPLC 9, HPLC methods have been reported for the estimation of Dasatinib. A new HPLC method was developed and validated as per ICH guidelines 17 for the estimation of Dasatinib in pharmaceutical formulations. Fig. 1: Chemical structure of Dasatinib Fig. 2: Linearity curve of Dasatinib MATERIALS AND METHODS Instrumentation To develop a high pressure liquid chromatographic method for quantitative estimation of Dasatinib using Waters HPLC system on Cosmicsil BDS C18 column (150 mm x 4.6 mm, 5μ) was used. The instrument is equipped with an auto sampler and DAD or UV detector. A 20 μl rheodyne injector port was used for injecting the samples. Data was analyzed by using Empower2 software. Chemicals and solvents The working standard of Dasatinib was provided as gift sample from Pharma Train, Hyderabad, India. The market formulation SPRYCEL tablets were procured from local market. HPLC grade water and methanol were purchased from E. Merck (India) Ltd, Mumbai, India. Triethyl amine and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd, Mumbai, India. Chromatographic conditions A mixture of phosphate buffer and solvent mixture of acetonitrile and methanol in the ratio of 50:50, v/v was found to be the most suitable mobile phase for ideal chromatographic separation of Dasatinib. The solvent mixture was filtered through a 0.45 μ membrane filter and sonicated before use. It was pumped through the column at a 8

3 flow rate of 1.0 ml/min. Injection volume was 10 µl and the column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 315 nm. The run time was set at 10 min. Preparation of phosphate buffer Added 4.0ml of triethylamine to 100ml water and adjusted the p H to 6.5±0.05 with dilute orthophosphoric acid. Preparation of solvent mixture and mobile phase Prepared a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively. Prepared, filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v respectively. Preparation of standard solution Accurately weighed and transferred the drug equivalent to 50 mg of Dasatinib (working standard) into 50ml volumetric flask. Added about 30ml of solvent mixture and sonicated to dissolve. Cooled the solution to room temperature and diluted to volume with solvent mixture. Transferred 1.0ml of the above solution into a 10ml volumetric flask and diluted to volume with mobile phase. Preparation of sample solution 10 tablets of marketed formulation was taken, weighed and finely powdered. Accurately weighed and transferred the drug powder equivalent to 50 mg of Dasatinib (working standard) into 50ml volumetric flask. Added about 30ml of solvent mixture, placed on orbital shaker for 15min and sonicated for 30min with occasional shakings. Cooled the solution to room temperature and diluted to volume with solvent mixture. Centrifuged the solution at 3000 rpm for 15min. Transferred 1ml of the above solution into a 10ml volumetric flask, dilutedto volume with mobile phase. Linearity Several aliquots of standard solution of Dasatinib was taken in different 10 ml volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Dasatinib were in the range of 20 to 60 µg/ml. Evaluation of the drug was performed with UV detector at 315 nm, peak area was recorded for all the peaks. The correlation coefficient value of Dasatinib was The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated. Limit of detection and limit of quantification The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solution using the developed HPLC method. The LOD for Dasatinib was found to be µg/mL. The LOQ for Dasatinib was found to be µg/ml. System suitability System suitability parameters like retention time, resolution, theoretical plates and tailing factor were calculated and compared with standard values. Accuracy The accuracy of the method was assessed by recovery study of Dasatinib in the dosage form at three concentration levels. A fixed amount of pre analyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The content Dasatinib per tablet was calculated. The mean recovery of was in the range Dasatinib of % that shows there is no interference from excipients and the lower values of RSD of assay indicate the method is accurate. Precision The precision was determined for Dasatinib in terms of intra-day and inter-day precision. For intra-day precision evaluation, a standard solution of fixed concentration was injected at various time intervals and %RSD for 9

4 Dasatinib was calculated and was found to be 0.80% (limit %RSD < 2.0%). In addition, the inter-day precision was studied by injecting the same concentration of standard solution on consecutive days and the %RSD for Dasatinib was calculated and was found to be 0.78% (limit %RSD < 2.0%). Ruggedness and robustness The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types. Robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the HPLC method so developed is rugged and robust. Assay 10 µl of each standard and sample solution were injected and from the peak area of Dasatinib, amount of each drug in samples were computed. The result of assay undertaken yielded 100.1% of label claim of Dasatinib. RESULTS AND DISCUSSION The HPLC procedure was optimized with a view to develop an accurate method in tablet dosage form using Cosmicsil BDS C18 column (150 x 4.6 mm, 5 μ) in isocratic mode with mobile phase composition of triethylamine buffer and mixture of acetonitrile: methanol (50:50, v/v) in the ratio of 50:50, v/v and ph adjusted to 6.5 with orthophosphoric acid. The use of this mobile phase resulted in peak with good shape and resolution. The flow rate was 1.0 ml/min and the drug component was measured with UV detector at 315 nm. The results of optimized HPLC conditions were shown in Table 1. The method was linear in the range of 20 to 60 µg/ml for Dasatinib with correlation coefficient of The linearity results were shown in Table 2 and the linearity curve was shown in Fig. 2. The % recovery was found to be 100.1% for Dasatinib, which indicate the method is accurate. The results of recovery studies were shown in Table 3. The %RSD for intra-day precision and inter-day precision for Dasatinib were found to be 0.80 and 0.74, which indicate the method is precise. The results of precision studies were shown in Table 4. Fig. 3: Typical chromatogram of Dasatinib Table 1: Optimized chromatographic conditions of Dasatinib Parameter Condition Mobile phase Buffer: (Acetonitrile:methanol (50:50, v/v)) 50:50, v/v ph 6.5 Diluent Mobile phase Column Cosmicsil BDS C18 column (150 mm x4.6 mm, 5μ) Column temperature Ambient Wave length 315nm Injection volume 10 µl Flow rate 1.0 ml/min Run time 10 min Retention time min 10

5 Table 2: Linearity results of Dasatinib Concentration in μg/ml Area Table 3: Recovery results of Dasatinib Level Amount added Amount found % Recovery Mean recovery 50% % 100% % % 150% % Table 4: Precision studies of Dasatinib Concentration (µg/ml) Intra-day precision (%RSD) Inter-day precision (%RSD) Table 5: Summary of system suitability and validation parameters of Dasatinib Parameter Results Linearity range (μg/ml) Correlation coefficient Theoretical plates (N) 7620 Tailing factor 1.24 LOD (μg/ml) LOQ (μg/ml) Table 6: Assay results of Dasatinib Formulation Label claim Amount found %Assay SPRYCEL 50 mg mg % The retention time of Dasatinib was 6.46 min. The number of theoretical plates was 7620 and tailing factor was 1.24 for Dasatinib, which indicates efficient performance of the column. The limit of detection and limit of quantification for Dasatinib were found to be µg/ml and µg/ml, which indicate the sensitivity of the method. The summary of system suitability parameters and validation parameters were shown in Table 5. Validated method was applied for the determination of Dasatinib in commercial formulations. The %assay was found to be 100.1% for Dasatinib and the assay results were shown in Table 6. Typical chromatogram of drug Dasatinib was shown in Fig. 3. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in the formulation did not interfere with the estimation of the drug by the proposed HPLC method. CONCLUSION A simple, precise, selective and sensitive RP-HPLC method with UV detection for Dasatinib was developed and validated. This method will be useful for the easy and quick estimation of Dasatinib with almost no interferences in bulk and pharmaceutical dosage forms. REFERENCES:

6 3. Sandra R, Gillian MM, Martin C and Robert OC. Development of a highperformance liquid chromatographicmass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors Lapatinib and Dasatinib. J Chromatogr B. 2009; 877(31): Silvia DF, Antonio DA, Francesca DM, Elisa P, Lorena B, Marco S, Marco S, Silvia R, Giuseppe S, Francesco DC and Giovanni DP. New HPLC-MS method for the simultaneous quantification of the antileukemia drugs Imatinib, Dasatinib, and Nilotinib in human plasma. J Chromatogr B. 2009; 877(18-19): A. D Avolio, M. Simiele, M. Siccardi, L. Baietto, M. Sciandra, S. Bonora, G. DiPerri, HPLC MS method for the quantification of nine anti-hiv drugs from dry plasma spot on glass filter and their long term stability in different conditions. Pharm. Biomed. Anal. 52 (2010) Antonio DA, Marco S, Silvia DF, Alessandra A, Lorena B, Jessica C, Carmen F, Giuseppe S, Francesco DC and Giovanni DP. HPLC-MS method for the simultaneous quantification of the antileukemia drugs Imatinib, Dasatinib and Nilotinib in human peripheral blood mononuclear cell (PBMC). J Pharm Biomed Anal. 2012;59: Haouala A, Zanolari B, Rochat B, Montemurro M, Zaman K, Duchosal MA, Ris HB, Leyvraz S, Widmer N and Decosterd LA. Therapeutic drug monitoring of the new targeted anticancer agents Imatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib and Lapatinib by LC tandem mass spectrometry. J Chromatogr B. 2009; 877(22): Michael TF, Shruti A, Dara H, Michael L, Steve U, Linda K and Bruce S. A validated LC-MS/MS assay for the simultaneous determination of the anti-leukemic agent Dasatinib and two pharmacologically active metabolites in human plasma: application to a clinical pharmacokinetic study. J Pharm Biomed Anal. 2012;58: Eva K, Jurij T, Tadej P and Albin K.Simultaneous measurement ofimatinib, Nilotinib and Dasatinib in Dried blood spot by ultra-highperformance liquid chromatography tandem mass spectrometry. JChromatogr B. 2012; 903: Elisa P, Silvia DF, Francesca DM, Carmen F, Stefano U, Giovanna Sand Francesco DC. A new HPLC-UV validated method for therapeutic drug monitoring of tyrosine kinase inhibitorsin leukemic patients. J ChromatogrSci. 2011; 49: Arun Kumar K, Ananta Rao B,Yaswanth A, Dayananda Chary P,Shanth Kumar S and Navya A. Development and validation of RPHPLCmethod for estimation of Dasatinib in bulk and its pharmaceutical formulation. Am JPharm Tech Res. 2012; 2(4): Mohammed G. Kassem, Essam Ezzeldin, Hesham M. Korashy, Gamal A.E. Mostafa High-performance liquid chromatographic method for the determination of Dasatinib in rabbit plasma using fluorescence detection and its application to a pharmacokinetic study Journal of Chromatography B.2013; 939: Thulase Nadh Reddy Dodda, Regalla Narasimha Reddy, Meghana Reddy Regalla. method development and validation of stability indicating rp--hplc method for the estimation of dasatinib in tablet dosage form. Indo American Journal of Pharmaceutical Research, (12) A. Sreedevi and A. Lakshmana Rao. Development and validation of novel HPLC method for the estimation of dasatinib in bulk and pharmaceutical dosage forms. International journal of research in pharmacy and chemistry. 2013, 3(3); Bandi Ramachandra, N.V.S.Naidu, Chandra K Sekhar. Validation of RP-HPLC Method for Estimation of Dasatinib in Bulk and its Pharmaceutical Dosage Forms. International Journal of Pharmacy and Biological Sciences. 2014; 4(1): Alagar Raja. M, Swapna. M, Shirisha. V, David Banji, Rao. K N V, Selva Kumar. D. RP-HPLC method for estimation of dasatinib in active pharmaceutical ingredient and pharmaceutical dosage form as per ICH guidelines. Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 2015; 3(3): ICH Harmonised Tripartite Guideline, Q2(R1), Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonisation, Geneva. 2005:

DEVELOPMENT AND VALIDATION OF NOVEL HPLC METHOD FOR THE ESTIMATION OF DASATINIB IN BULK AND PHARMACEUTICAL DOSAGE FORMS

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY Available online at www.ijrpc.com Research Article DEVELOPMENT AND VALIDATION OF NOVEL HPLC METHOD FOR THE ESTIMATION OF DASATINIB IN BULK AND